DNA Repair

Development of a High-Content High-Throughput Screening Assay for the Discovery of ATM Signaling Inhibitors.

J Biomol Screen. 2012 Aug;17(7):912-20

Authors: Bardelle C, Boros J

Abstract
The genome is constantly exposed to DNA damage agents, leading up to as many as 1 million individual lesions per cell per day. Cells have developed a variety of DNA damage repair (DDR) mechanisms to respond to harmful effects of DNA damage. Failure to repair the damaged DNA causes genomic instability and, as a result, leads to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers, thus providing a great opportunity for cancer therapy by developing anticancer agents that work by synthetic lethality-based mechanisms or enhancing the clinical efficacy of radiotherapy and existing chemotherapies. Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to DNA double-strand breaks. Ionizing radiation causes double-strand breaks and induces rapid ATM autophosphorylation on serine 1981 that initiates ATM kinase activity. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage-response pathways, most notably cell-cycle checkpoints. We describe here the development and validation of a high-throughput imaging assay measuring levels of phospho-ATM Ser1981 in HT29 cells after exposure to ionizing radiation. We also examined activation of downstream ATM effectors and checked specificity of the endpoint using known inhibitors of DNA repair pathways.

PMID: 22653913 [PubMed - in process]

Mycobacterium tuberculosis RecG Binds and Unwinds Model DNA Substrates with a Preference for Holliday Junctions.

Microbiology. 2012 May 24;

Authors: Zegeye ED, Balasingham SV, Laerdahl JK, Homberset H, Tønjum T

Abstract
The RecG enzyme, a superfamily 2 helicase, is present in nearly all bacteria. Here, we report for the first time that the recG gene was also found to be present in the genomes of most vascular plants as well as in green algae, but was not found in other eukaryotes or the archaea. The precise function of RecG is poorly understood, even though ample evidence shows that it plays critical roles in DNA repair, recombination and replication. We further demonstrate that the Mycobacterium tuberculosis RecG (RecGMtb) DNA binding activity had a broad substrate specificity, while it only unwound branched-DNA substrates such as Holliday junctions (HJ), replication forks, D-loops and R-loops, with a strong preference for the HJ as a helicase substrate. In addition, RecGMtb preferentially bound relatively long (¡Ý40-nt) ssDNA, exhibiting a higher affinity towards the homopolymeric nucleotides poly(dT), poly(dG) or poly(dC) than for poly(dA). RecGMtb helicase activity was supported by hydrolysis of ATP or dATP in the presence of Mg2+, Mn2+, Cu2+ or Fe2+. Like its E. coli ortholog, RecGMtb is also a strictly DNA-dependent ATPase.

PMID: 22628485 [PubMed - as supplied by publisher]

Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB.

PLoS One. 2012;7(5):e36960

Authors: Balasingham SV, Zegeye ED, Homberset H, Rossi ML, Laerdahl JK, Bohr VA, Tønjum T

Abstract
XPB, also known as ERCC3 and RAD25, is a 3'?5' DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3'?5' DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg(2+)/Mn(2+). Consistent with the 3'?5' polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3' overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3' DNA tail, it was not active on substrates containing a 3' RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB.

PMID: 22615856 [PubMed - in process]

Molecular mechanisms of temozolomide resistance in glioblastoma multiforme.

Expert Rev Anticancer Ther. 2012 May;12(5):635-42

Authors: Johannessen TC, Bjerkvig R

Abstract
Glioblastoma multiforme (GBM; WHO astrocytoma grade IV) is considered incurable owing to its inherently profound resistance towards current standards of therapy. Considerable effort is being devoted to identifying the molecular basis of temozolomide resistance in GBMs and exploring novel therapeutic regimens that may improve overall survival. Several independent DNA repair mechanisms that normally safeguard genome integrity can facilitate drug resistance and cancer cell survival by removing chemotherapy-induced DNA adducts. Furthermore, subpopulations of cancer stem-like cells have been implicated in the treatment resistance of several malignancies including GBMs. Thus, a growing number of molecular mechanisms contributing to temozolomide resistance are being uncovered in preclinical studies and, consequently, we are being presented with a broad range of potentially novel targets for therapy. A substantial future challenge is to successfully exploit the increasing molecular knowledge contributing to temozolomide resistance in robust clinical trials and to ultimately improve overall survival for GBM patients.

PMID: 22594898 [PubMed - in process]